Enzyme-Mimics for Sensitive and Selective Steroid Metabolite Detection

Abstract

We present an enzyme-like functional polymer that recognizes nonelectroactive targets and catalyzes their redox reactions for simple, selective steroid metabolite detection. Measuring steroid metabolites, such as cortisol, has been widely adopted to diagnose stress and chronic diseases. Conventional detection method based on competitive immunoassay requires time-consuming labeling processes for signal transduction and unstable biological receptors for biorecognition yet with limited selectivity. Inspired by natural enzymes' target specificity and catalytic nature, we report an enzyme-mimic using electrocatalytic molecularly imprinted polymers (EC-MIP) to achieve label-free, external redox reagent-free, sensitive, and selective electrochemical detection of cortisol. The EC-MIP sensor contains molecularly imprinted cavities for specific cortisol binding and embedded copper phthalocyanine tetrasulfonate (CuPcTS) for electrocatalytic reduction of the ketones on the captured cortisol into alcohols. The direct sensing approach resolves the intrinsic limitations of conventional MIP-based sensors, most notably the use of external redox probes and weak sensing signals. The sensor exhibited a detection limit of 181 pM with significantly enhanced selectivity using a differential sensing mechanism. The new enzyme-like sensor can be modified to detect other targets, offering a simple, robust approach to future health monitoring technologies.

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“…Here, σ I denotes the standard deviation of the sensing signal for blank samples, and Δ I is a function of target concentration c fitted by the isotherm model in eq . The derivation is detailed in our previous report . The LOD values obtained for cortisol redox MIP and DHEA redox MIP are 115 and 390 pM, respectively.…”

“…A smaller value of m indicates a broader distribution of The derivation is detailed in our previous report. 29 The LOD values obtained for cortisol redox MIP and DHEA redox MIP are 115 and 390 respectively. These values demonstrate a remarkable improvement compared to other reported redox reagent-free steroid MIP sensors 25,54 and are comparable to the performance of our previous electrocatalytic MIP sensors.…”

“…These values demonstrate a remarkable improvement compared to other reported redox reagent-free steroid MIP sensors 25,54 and are comparable to the performance of our previous electrocatalytic MIP sensors. 29 A solution mixture containing various combinations of cortisol and DHEA concentrations was used to evaluate the cross-sensitivity of the integrated sensor. Both cortisol and DHEA redox MIP sensors demonstrated successful target detection while effectively discriminating the presence of nontargets.…”

“…This approach strategically anchors the redox substances near the binding cavities, enabling sensitive response while offering straightforward, reliable, and cost-effective detection capabilities that are well-suited for on-site and frequent testing. Our prior study reported an electrocatalytic MIP for sensitive cortisol detection through direct reduction of cortisol, which requires a relatively large potential (−1 V vs Ag/AgCl) as determined by the catalyst . The redox MIP presented in this study offers an alternative approach for detecting steroid hormones utilizing a lower voltage in amperometry measurements governed by the redox-active polymer.…”

“…Our prior study reported an electrocatalytic MIP for sensitive cortisol detection through direct reduction of cortisol, which requires a relatively large potential (−1 V vs Ag/AgCl) as determined by the catalyst. 29 The redox MIP presented in this study offers an alternative approach for detecting steroid hormones utilizing a lower voltage in amperometry measurements governed by the redox-active polymer.…”